Pseudoxanthomonas Wuyuanensis Sp. Nov., Isolated from Saline-Alkali Soil

%paper no. ije056796 charlesworth ref: ije056796& New Taxa - Proteobacteria Open Access at HighWire International Journal of Systematic and Evolutionary Microbiology (2014), 64, 000–000 DOI 10.1099/ijs.0.056796-0

Pseudoxanthomonas wuyuanensis sp. nov., isolated from saline-alkali soil

Dai Li,1 Huancheng Pang,2 Licui Sun,3 Jinping Fan,1 Yuyi Li2 and Jianli Zhang1

Correspondence 1School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China Jianli Zhang 2Institute of Agri-resources and Regional Planning, CAAS, Beijing 100081, PR China [email protected] 3Department of Nutrition and Metabolism, National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, PR China

A bacterium, designated XC21-2T, was isolated from a saline-alkaline soil sample from China. Cells were Gram-stain-negative, rod-shaped and motile and grew optimally at 35–37 6C, pH 6.0–7.0 and in the presence of 0.5 % (w/v) NaCl. Growth occurred in the range pH 5.5–9.0

and in the presence of up to 4 % (w/v) NaCl. The major cellular fatty acids were iso-C15 : 0,

iso-C16 : 0 and iso-C17 : 1v9c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an uncharacterized amino-group-containing polar lipid. The major quinone was ubiquinone 8 (Q-8) and the G+C content of the genomic DNA was 66.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain XC21-2T formed a tight phylogenetic lineage with Pseudoxanthomonas dokdonensis DS-16T within the genus Pseudoxanthomonas and was most closely related to P. dokdonensis DS-16T and P. mexicana AMX 26BT, with 97.9 and 97.5 % 16S rRNA gene sequence similarity, respectively. On the basis of the unique physiological profile of the isolate and its phylogenetic divergence from known species, strain XC21-2T represents a novel species within the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas wuyuanensis sp. nov. is proposed. The type strain is XC21-2T (5CGMCC 1.10978T5KCTC 23877T).

The genus Pseudoxanthomonas was described by Finkmann Strain XC21-2T was isolated from a saline-alkaline soil sample et al. (2000) for Pseudoxanthomonas broegbernensis, repre- [pH 8.5; salt content 0.4 % (w/w)] collected from a sun- sented by a strain isolated from an experimental biofilter flower plot in Wuyuan county (40u 469 300 N107u 359 700 E) ; supplied with the waste gas of an animal-rendering plant. in Inner Mongolia in north-west China. The soil sample was At the time of writing, the genus contained 14 species, which suspended in distilled water and then the sus- were isolated from a biofilter (Finkmann et al., 2000), a hot pension was diluted with sterile distilled water and incu- spring (Chen et al., 2002), soils (Thierry et al., 2004; Chang bated at 35 uC on Petri dishes containing nutrient agar (3 g et al., 2005; Yang et al., 2005; Harada et al., 2006; Yoo et al., beef extract, 5 g peptone, 5 g NaCl, 1 l distilled water; 2007; Young et al., 2007; Lee et al., 2008; Kumari et al., 2011) pH 7.6) for 3 days. Purification was obtained by streaking and compost (Weon et al., 2006). Members of the genus single colonies on Petri dishes filled with trypticase soy agar Pseudoxanthomonas are Gram-stain-negative rods and con- (TSA; Difco); purified strains were maintained on TSA at tain ubiquinone 8 (Q-8) as the predominant isoprenoid 4 uC and as glycerol suspensions (20 %, v/v) at 220 uC. quinone. The genomic DNA G+C content is 65–70 mol%. Cells for morphological, physiological and biochemical The cellular fatty acids are of the iso-/anteiso-branched type, characterization of strain XC21-2T and reference strains with iso-C normally predominating and iso-C 3-OH 15 : 0 13 : 0 were prepared by routine cultivation on TSA at 35 uC. Cell always absent with one exception; Pseudoxanthomonas morphology and motility were deduced from direct spadix contains iso-C 3-OH (Thierry et al., 2004; Lee 13 : 0 observations of fresh cultures using light microscopy et al., 2008). (BH-2; Olympus) and transmission electron microscopy (JEM-1400; JEOL). Growth at 4–45 uC, pH 5.0–10.0 (in The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene increments of 0.5 pH units) and 0–5 % (w/v) NaCl (in sequence of strain XC21-2T is JN247803. 1.0 % increments) was examined on TSA. Gram staining Two supplementary figures are available with the online version of this was performed as described by Gerhardt et al. (1981). paper. Activities of catalase and oxidase and hydrolysis of Tween

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D. Li and others

80, casein, gelatin, starch and urea were investigated as as the spraying reagent for detection of total lipids (Gupta described by Cowan & Steel (1965). Hydrolysis of aesculin et al., 2009; Sharma et al., 2010; Nigam et al., 2010). The T and nitrate reduction were studied as described by La´nyi major fatty acids of strain XC21-2 (.10 %) were iso-C15 : 0, (1987). Sensitivity to antibiotics was tested on TSA plates iso-C16 : 0 and iso-C17 : 1v9c; methyl-branched fatty acids using antibiotic discs. Additional enzyme activities and corresponded to about 80 % of the total cellular fatty acids biochemical characteristics were determined by using the (Table 2). The absence of the fatty acid iso-C13 : 0 3-OH is a API ZYM (37 uC; 4 h) and API 20NE (30 uC; 24 h) kits, as general property of species of the genus Pseudoxanthomonas, described by the manufacturer (bioMe´rieux). The results T but strain XC21-2 had iso-C13 : 0 3-OH in trace amounts, as of phenotypic and physiological characterization of strain T T reported for P. broegbernensis DSM 12573 , P. suwonensis XC21-2 and type strains of related species are given in T T 4M1 and P. spadix IMMIB AFH-5 . Q-8 was the predo- Table 1 and the species description. minant ubiquinone of strain XC21-2T, in agreement with Cells for analysis of cellular fatty acids were cultivated the characteristics of species of the genus Pseudoxanthomonas. on TSA at 30 uC for 48 h. Fatty acid methyl esters were Major polar lipids present in strain XC21-2T were extracted and prepared according to the standard protocol phosphatidylethanolamine, phosphatidylglycerol, dipho- of the MIDI/Hewlett Packard Microbial Identification sphatidylglycerol and an uncharacterized amino-group- System (Sasser, 1990). Isoprenoid quinones were extracted containing polar lipid (Fig. S1, available in IJSEM according to the method of Komagata & Suzuki (1987) and Online). The major polar lipids of strain XC21-2T were analysed using reversed-phase HPLC and a YMC ODS-A the same as those of Pseudoxanthomonas indica and (25064.6 mm) column. Polar lipid analysis was performed Pseudoxanthomonas sacheonensis. The DNA G+C content by two-dimensional TLC using molybdatophosphoric acid of strain XC21-2T was analysed according to the methods

Table 1. Phenotypic characteristics that differentiate strain XC21-2T from type strains of phylogenetically related species

Strains: 1, XC21-2T;2,P. dokdonensis DS-16T;3,P. mexicana AMX 26BT; 4, P. daejeonensis TR6-08T (data from Yang et al., 2005); 5, P. > broegbernensis B1616/1T (Thierry et al., 2004); 6, P. japonensis 12-3T (Thierry et al., 2004); 7, P. koreensis T7-09T (Yang et al., 2005); 8, P. suwonensis T T 4M1 (Weon et al., 2006); 9, P. yeongjuensis GR12-1 (Yoo et al., 2007). +, Positive test or growth; 2, negative test or no growth; V, variable growth between duplicates; W, weak response; ND, no data available; ONPG, o-nitrophenyl b-D-galactopyranoside. All strains are negative for assimilation of D-mannitol, D-gluconate, caprate, adipate and phenylacetate. Data were obtained from this study unless indicated.

Characteristic 1 2 3 4 5 6 7 8 9

Motility and flagellum + 2 ++ ++2 ++ Optimum temperature (uC) 35–37 30 30–37 30 30 30–37 30 30 28 Growth at: 10 uC ++ + W ++ W ++ 42 uC W 22 2 2 2 2 + 2 Catalase ++ W ++2 +++ Arginine dihydrolase 22 2 2 2 2 + 22 Urease 22 2 2 2 2 + 22 Hydrolysis of: Casein + 2 ++ 2 ++ 2 + Gelatin ++ + 22+ 22+ Tween 80 ++ + ND ++ND 2 ND Starch 22 VND 22ND 2 ND Aesculin ++ + + + + 2 ++ ONPG + 22 +++2 ++ Assimilation of: D-Glucose ++ + + + + 2 ++ L-Arabinose + 22 ++22 + 2 D-Mannose 2 ++ 2 + 22 22 N-Acetylglucosamine ++ + + + + 2 ++ Malate + 22 2 2+ (L)* 222 Citrate 22 2 2 + 22 22 Nitrate reduction 2 + 22 222 + 2 DNA G+C content (mol%) 66.2 65.1aD 67.8±2b 68.7±0.4 66.5±0.8 65.2±1 69.5±0.5 67.5±0.9 63.4

*Positive for assimilation of L-malate. DData taken from: a, Yoon et al. (2006); b, Thierry et al. (2004).

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Pseudoxanthomonas wuyuanensis sp. nov. of Tamaoka & Komagata (1984) and was determined to be calculated according to Kimura (1980). DNA–DNA 66.2 mol%, which is similar to results reported for other hybridization was carried out to evaluate genomic DNA members of the genus Pseudoxanthomonas (Table 1). relatedness between strain XC21-2T and its closest neigh- bours (P. dokdonensis DS-16T and P. mexicana AMX 26BT) < Genomic DNA was prepared from strain XC21-2T using a thermal denaturation procedure (De Ley et al., according to Yoon et al. (1996). The 16S rRNA gene was 1970; Huss et al., 1983) with a UV-1206 spectrophotometer amplified by PCR as described by Yoon et al. (1998), and (Shimadzu) fitted with a TB-85 thermal bath and standard sequencing of the PCR product was performed using software (Jahnke, 1992). Hybridization was performed with the method of Yoon et al. (2003). The 16S rRNA gene five replications; the highest and lowest values obtained sequences of related taxa were obtained from GenBank. were excluded and the means of the remaining three values Multiple alignment with sequences of closely related are quoted as DNA–DNA relatedness values. species was performed using the CLUSTAL_X 1.8 software (Thompson et al., 1997). Phylogenetic dendrograms were The 16S rRNA gene sequence of strain XC21-2T consisted reconstructed using the neighbour-joining (Saitou & Nei, of 1426 nt and sequence analysis showed that the isolate 1987), maximum-parsimony (Kluge & Farris, 1969) and belonged to the genus Pseudoxanthomonas. Phylogene- T T maximum-likelihood (Felsenstein, 1981) methods in MEGA tically, strain XC21-2 is related to P. dokdonensis DS-16 , version 5 (Tamura et al., 2011), with bootstrap values with a bootstrap value of 52 % (Fig. 1). Pairwise 16S rRNA based on 1000 replications. Evolutionary distances were gene sequence similarity between strain XC21-2T and

Table 2. Cellular fatty acid profiles of strain XC21-2T and related type strains

Strains: 1, XC21-2T;2,P. dokdonensis DS-16T;3,P. mexicana AMX 26BT;4,P. daejeonensis TR6-08T (data from Yang et al., 2005); 5, P. broegbernensis B1616/1T (Thierry et al., 2004); 6, P. japonensis 12-3T (Thierry et al., 2004); 7, P. koreensis T7-09T (Yang et al., 2005); 8, P. suwonensis T T 4M1 (Weon et al., 2006); 9, P. yeongjuensis GR12-1 (Yoo et al., 2007). Values are percentages of total fatty acids. TR, Trace, corresponding arbitrarily to less than 0.34 % of total cellular fatty acids; 2, not detected. Data were obtained from this study unless indicated. The three strains tested in this study were grown under the same conditions (30 uC, 48 h).

Fatty acid 1 2 3 4 5 6 7 8 9

Saturated

C14 : 0 TR 2 0.7 2 1.8 0.4 2 0.4 2 C16 : 0 0.7 2.1 2.1 1.2 10.3 0.7 2 1.0 2.3 Unsaturated

C16 : 1cis9 2222 9.4 2222 C16 : 1v9c (C16 : 1cis7) 2226.7 1.0 2 2.3 0.4 2 C16 : 1v7c alcohol 5.9 0.7 22 2 2221.6 C18 : 1v7c 0.5 1.3 22 2 2222 Methyl-branched

iso-C11 : 0 5.5 4.9 5.8 2 5.4 4.7 2 7.4 5.5 iso-C14 : 0 1.1 1.8 2.9 2.2 0.5 2.4 2 2.9 1.4 iso-C15 : 1 F 221.8 2 1.5 1.5 2 1.3 2 iso-C15 : 0 27.4 22.3 36.2 40.5 26.7 33.0 46.2 30.8 32.0 anteiso-C15 : 0 2.7 3.7 2.7 6.9 14.1 3.9 1.0 13.2 4.4 iso-C16 : 1 H 0.9 2 4.3 224.1 2 0.4 2 iso-C16 : 0 15.4 25.4 9.5 8.0 2.6 12.7 2 11.1 13.3 iso-C17 : 0 3.4 4.2 3.4 3.6 3.6 3.5 7.8 2.7 6.4 anteiso-C17 : 0 1.6 2.3 0.4 2 0.9 0.6 2 1.1 1.5 iso-C15 : 1 at 5 4.5 222 2 2221.0 iso-C17 : 1v9c (iso-C17 : 1cis7) 17.2 10.8 16.8 20.3 10.7 19.9 24.4 10.6 14.6 Branched-chain hydroxy

iso-C11 : 0 3-OH 5.0 4.3 6.2 2 5.9 5.2 2 7.0 7.7 iso-C12 : 0 3-OH 1.1 1.2 TR 2 TR TR 2 1.1 2 iso-C13 : 0 3-OH TR 222 TR 22TR 2 Unresolved

iso-C16 : 1 I/C14 : 0 3-OH 1.1 222 2 2222 C16 : 1v7c/iso-C15 : 0 2-OH 2.3 8.1 3.2* 2 10.9* 4.5* 2 4.1 4.0

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related taxa was determined by using the EzTaxon server Description of Pseudoxanthomonas wuyuanensis (Chun et al., 2007). The closest relatives in terms of pairwise sp. nov. 16S rRNA gene sequence similarity were P. dokdonensis Pseudoxanthomonas wuyuanensis (wu.yua.nen9sis. N.L. fem. DS-16T (97.9 %), P. mexicana AMX 26BT (97.5 %), P. daejeonensis TR6-08T (97.3 %), P. broegbernensis B1616/1T adj. wuyuanensis of Wuyuan, where the type strain was (97.0 %), P. japonensis 12-3T (97.0 %) and the type strains isolated). of the other species of the genus Pseudoxanthomonas Cells are Gram-stain-negative, aerobic, motile rods (96.7–94.2 %). The mean DNA–DNA hybridization found (2.0060.65 mm) with a single polar flagellum. Colonies T between XC21-2 and these five type strains was 27.3 % (P. on TSA are circular, convex, sticky, glistening and dark = dokdonensis), 20.6 % (P. mexicana), 21.8 % (P. daejeonensis), yellow and 1.5–2.5 mm in diameter after 3 days of 19.8 % (P. broegbernensis) and 19.4 % (P. japonensis), values incubation at 35 uC. Optimal growth occurs at 35–37 uC, well below the 50 % cut-off point recommended by Wayne pH 6.0–7.0 and in the presence of 0.5 % (w/v) NaCl. et al. (1987) for the delineation of genomic species. Growth occurs at pH 5.5–9.0 and in the presence of up to Morphological and chemotaxonomic characteristics and 4 % (w/v) NaCl. Catalase- and oxidase-positive. Nitrate is 16S rRNA gene sequence comparisons of strain XC21- not reduced to nitrite. Hydrolyses casein, gelatin, Tween 2T suggest the assignment of the isolate to the genus 80, aesculin and ONPG, but not urea or starch. Negative Pseudoxanthomonas. Strain XC21-2T could be separated for indole production and arginine dihydrolase activity. from its closest relative, P. dokdonensis DS-16T, on the basis Assimilates D-glucose, L-arabinose, N-acetylglucosamine of motility and the presence of a flagellum (Fig. S2), nitrate and malate, but does not assimilate D-mannose, citrate, D- reduction, growth at 35–37 uC, hydrolysis of casein and o- mannitol, D-gluconate, caprate, adipate or phenylacet- nitrophenyl b-D-galactopyranoside (ONPG), assimilation ate. Produces alkaline phosphatase, esterase (C4), esterase of L-arabinose and D-mannose and enzyme and fatty acid lipase (C8), leucine arylamidase, trypsin, acid phos- profiles. The unique physiological profile of strain XC21-2T phatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, (Table 1) and its phylogenetic divergence from related type b-galactosidase, b-glucosidase, N-acetyl-b-glucosaminidase strains (Fig. 1) demonstrated that the strain represents a and a-mannosidase, but not lipase (C14), valine arylamidase, separate species within the genus Pseudoxanthomonas. cystine arylamidase, a-chymotrypsin, b-glucuronidase, a- On the basis of these data, strain XC21-2T should be glucosidase or a-fucosidase. Susceptible to gentamicin, classified as the type strain of a novel species of the genus streptomycin, tetracycline, rifampicin, nalidixic acid, chlor- Pseudoxanthomonas,forwhichthenamePseudoxanthomonas amphenicol, neomycin, amikacin, doxycycline, ciprofloxacin, wuyuanensis sp. nov. is proposed. ofloxacin, gatifloxacin, tobramycin and polymyxin B. The

52 Pseudoxanthomonas daejeonensis JCM 21575T (AY550264) 0.005 89 Pseudoxanthomonas koreensis IAM 15116T (AY550263) 99 Pseudoxanthomonas kaohsiungensis BCRC 17375T (AY650027) Pseudoxanthomonas broegbernensis ATCC BAA-10T (AJ012231) Pseudoxanthomonas suwonensis DSM 17175T (AY927994) Pseudoxanthomonas taiwanensis ATCC BAA-404T (AF427039) Pseudoxanthomonas mexicana ATCC 700993T (AF273082) 62 100 Pseudoxanthomonas japonensis CIP 107388T (AB008507) 52 Pseudoxanthomonas wuyuanensis XC21-2T (JN247803) Pseudoxanthomonas dokdonensis CIP 108839T (DQ178977) 88 100 Pseudoxanthomonas yeongjuensis DSM 18204T (DQ438977) Pseudoxanthomonas sacheonensis DSM 19373T (EF575564) Pseudoxanthomonas kalamensis ATCC BAA-1031T (AY686710) 68 Pseudoxanthomonas spadix CCUG 53828T (AM418384) Pseudoxanthomonas indica CCM 7430T (EF424397) Lysobacter enzymogenes DSM 2043T (AJ298291)

Fig. 1. Phylogenetic dendrogram based on 16S rRNA gene sequences showing the position of strain XC21-2T within the genus Pseudoxanthomonas and members of related genera. The tree was reconstructed using the neighbour-joining method. Bootstrap values above 50 % are represented as percentages of 1000 replicates at branching points. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony and maximum-likelihood trees. Lysobacter enzymogenes DSM 2043T was used as an outgroup. Bar, 0.005 substitutions per nucleotide position.

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Pseudoxanthomonas wuyuanensis sp. nov. major isoprenoid quinone is Q-8. The major cellular fatty Huss, V. A., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from acids are iso-C15 : 0,iso-C16 : 0 and iso-C17 : 1v9c.Themajor polar lipids are phosphatidylethanolamine, phosphatidylgly- renaturation rates. Syst Appl Microbiol 4, 184–192. cerol, diphosphatidylglycerol and an uncharacterized amino- Jahnke, K.-D. (1992). Basic computer program for evaluation of group-containing polar lipid. spectroscopic DNA renaturation data from Gilford System 2600 spectrophotometer on a PC/XT/AT type personal computer. JMicrobiol The type strain, strain XC21-2T (5CGMCC 1.10978T5 Methods 15, 61–73. T KCTC 23877 ), was isolated from a saline-alkaline soil Kimura, M. (1980). A simple method for estimating evolutionary rates sample collected from Wuyuan county, Inner Mongolia, of base substitutions through comparative studies of nucleotide China. The DNA G+C content of the type strain is sequences. J Mol Evol 16, 111–120. 66.2 mol%. The species description is based on a single Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the strain and hence serves as a description of the type strain. evolution of anurans. Syst Zool 18, 1–32. Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207. Acknowledgements Kumari, K., Sharma, P., Tyagi, K. & Lal, R. (2011). Pseudoxanthomonas This research was supported by the National Natural Science indica sp. nov., isolated from a hexachlorocyclohexane dumpsite. Int Foundation of China (NSFC, grant nos 31070002 and 31000692) J Syst Evol Microbiol 61, 2107–2111. and by the Special Fund for Public Welfare Industrial (Agriculture) La´ nyi, B. (1987). Classical and rapid identification methods for Research of China (grant no. 200903001). Transmission electron medically important bacteria. Methods Microbiol 19, 1–67. microscopy was carried out by the instrument centre of the Institute Lee, D. S., Ryu, S. H., Hwang, H. W., Kim, Y.-J., Park, M., Lee, J. R., of Microbiology, Chinese Academy of Sciences. The authors are Lee, S.-S. & Jeon, C. O. (2008). Pseudoxanthomonas sacheonensis sp. grateful to Dr Yafang Tan for carrying out quantitative fatty acid nov., isolated from BTEX-contaminated soil in Korea, transfer of analysis on strain XC21-2T. Stenotrophomonas dokdonensis Yoon et al. 2006 to the genus Pseudoxanthomonas as Pseudoxanthomonas dokdonensis comb. nov. and emended description of the genus Pseudoxanthomonas. Int J Syst References Evol Microbiol 58, 2235–2240. Chang, J. S., Chou, C. L., Lin, G. H., Sheu, S. Y. & Chen, W. M. (2005). Nigam, A., Jit, S. & Lal, R. (2010). Sphingomonas histidinilytica sp. Pseudoxanthomonas kaohsiungensis, sp. nov., a novel bacterium nov., isolated from a hexachlorocyclohexane dump site. Int J Syst Evol isolated from oil-polluted site produces extracellular surface activity. Microbiol 60, 1038–1043. Syst Appl Microbiol 28, 137–144. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Chen, M.-Y., Tsay, S.-S., Chen, K.-Y., Shi, Y.-C., Lin, Y.-T. & Lin, G.-H. method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– (2002). Pseudoxanthomonas taiwanensis sp. nov., a novel thermo- 425. philic, N2O-producing species isolated from hot springs. Int J Syst Sasser, M. (1990). Identification of bacteria by gas chromatography of Evol Microbiol 52, 2155–2161. cellular fatty acids, Technical Note 101. Newark, DE: MIDI, Inc. Chun, J., Lee, J. H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W. Sharma, P., Verma, M., Bala, K., Nigam, A. & Lal, R. (2010). (2007). EzTaxon: a web-based tool for the identification of Sphingopyxis ummariensis sp. nov., isolated from a hexachlorocyclo- prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst hexane dump site. Int J Syst Evol Microbiol 60, 780–784. Evol Microbiol 57, 2259–2261. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of composition by reversed-phase high-performance liquid chromato- Medical Bacteria. London: Cambridge University Press. graphy. FEMS Microbiol Lett 25, 125–128. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, measurement of DNA hybridization from renaturation rates. Eur S. (2011). MEGA5: molecular evolutionary genetics analysis using J Biochem 12, 133–142. maximum likelihood, evolutionary distance, and maximum par- Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a simony methods. Mol Biol Evol 28, 2731–2739. maximum likelihood approach. J Mol Evol 17, 368–376. Thierry, S., Macarie, H., Iizuka, T., Geissdo¨ rfer, W., Assih, E. A., Finkmann, W., Altendorf, K., Stackebrandt, E. & Lipski, A. (2000). Spanevello, M., Verhe, F., Thomas, P., Fudou, R. & other authors

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D. Li and others suwonensis sp. nov., isolated from cotton waste composts. Int J Syst Yoon, J.-H., Lee, S.-T. & Park, Y.-H. (1998). Inter- and intraspecific Evol Microbiol 56, 659–662. phylogenetic analysis of the genus Nocardioides and related taxa based Yang, D.-C., Im, W.-T., Kim, M. K. & Lee, S.-T. (2005). Pseudoxanthomonas on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194. koreensis sp. nov. and Pseudoxanthomonas daejeonensis sp. nov. Int Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. J Syst Evol Microbiol 55, 787–791. nov., isolated from jeotgal, a traditional Korean fermented seafood. Yoo, S.-H., Weon, H.-Y., Kim, B.-Y., Kim, J.-H., Baek, Y.-K., Kwon, S.-W., Int J Syst Evol Microbiol 53, 449–454. Go, S.-J. & Stackebrandt, E. (2007). Pseudoxanthomonas yeongjuensis Yoon, J. H., Kang, S. J., Oh, H. W. & Oh, T. K. (2006). Stenotrophomonas sp. nov., isolated from soil cultivated with Korean ginseng. Int J Syst dokdonensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, Evol Microbiol 57, 646–649. 1363–1367. Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Young, C. C., Ho, M.-J., Arun, A. B., Chen, W.-M., Lai, W.-A., Shen, F.-T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora Rekha, P. D. & Yassin, A. F. (2007). Pseudoxanthomonas spadix sp. nov., strains by the use of genomic DNA fragments and rRNA gene probes. isolated from oil-contaminated soil. Int J Syst Evol Microbiol 57, 1823– Int J Syst Bacteriol 46, 502–505. 1827.

6 International Journal of Systematic and Evolutionary Microbiology 64 Dear Authors,

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1 This longitude and latitude must be incorrect, as there are only 60 seconds in a minute of arc (so 70 cannot be correct). Please clarify whether the correct co-ordinates are ‘‘406 46.309 N 1076 35.709 E’’ or supply the correct longitude and latitude.

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